P U L K R A B E K ,
G R U N B E R G E R ,
A N D
W E I N S T E I N
Effects of the Ionic Environment on Modification of Yeast Tyrosine Transfer Ribonucleic Acid with &’-Acetoxy-2-acetylaminofluorenei Peter Pulkrabek,* Dezider Grunberger, 1 and I. Bernard Weinstein
ABSTRACT: The effects of Mga+ and K+ ions on modification of purified yeast tyrosine transfer ribonucleic acid with N acetoxy-2-acetylaminofluorene, a potent carcinogen which specifically binds to the 8 position of guanosine residues, were studied. In the presence of 3 mM Mg2+and 0.1 M K+ when the native form of tRNA is preserved, only 1.08 mol of the drug was bound/mol of tRNA. After pancreatic ribonuclease digestion of the modified tRNA, DEAE-cellulose chromatography of the resulting oligonucleotides, and Aminex A-6 separation of the component nucleosides, it was found that the major targets of the modification were two residues in the dihydrouridine loop, 2’-O-methylguanosine at position 18 and guanosine at position 19. In addition, a minor target was found in a dinucleotide probably derived from the anticodon
T
here is considerable evidence that tRNA possesses a highly organized three-dimensional structure which depends on the presence of divalent and/or univalent cations (Fresco et a/., 1966). Multiphase melting curves of individual purified tRNAs and other physicochemical measurements indicate that several “metastable” conformations can exist between tRNA in the “native” and “denatured” forms (Cole et a/., 1972; Cole and Crothers, 1972). Magnesium ions seem to be the most important factor in stabilization of the secondary and tertiary structures (Goldstein et a/., 1972), though they can be substituted to a considerable extent by other divalent cations, particularly Mn2+ and Ca2-, and to a lesser extent Zn2+ and Co2+(Fresco et d., 1966). The univalent cations K* and NaT can also stabilize the structure of tRNA; however, only a t much higher concentrations than magnesium ions (Fresco et al.,1966; Cole et a[.. 1972; Cole and Crothers, 1972). A partial renaturation of tRNA can also be achieved by polyamines (Tissikres, 1959; Mahler and Mehrotra, 1963). Chemical modification of tRNA appears to be a useful approach for providing information on the three-dimensional structure of tRNA and on certain structure-function relationships. N-Acetoxy-2-acetylaminofluorene, a derivative of a potent carcinogen N-2-acetylaminof-luorene, is a particularly iF r o m the Institute of Cancer Research and the Departments of Biochemistry and Medicine, College of Physicians and Surgeons, Columbia University, N e w York, New York 10032. Recciied D e c e d x r 3, 1973. This work was supported by U . S. Public Hcnlth Servicc Grant CA-02332. $ Scholar of Leukemia Society ofAmerica. 1 Abbreviations used are: AcNH-Fln, ,~-2-acetylarniliofluoreile; AcN(OAc)Fln, ,~-acetoxv-2-ncet!.laniinoRuorene; tRNA’\’, tRNA specific for tyrosine; tyros>I-tRNAT3’, the same t R N A acqlatctl with tyrosine: BD-cellulose, benzoylareci clicthylaiiiiiioethyl-cellulosc: G* denote5 guanosine residues modified with N-2-acetylaminofluorcne. Stantlard abbrcviationr \\cri) uscd according to I U P A C - I U B Com9, mission on Biochcmicel Nomencloturc (CBN) (1970), Biochcr?iistr~~ 4022.
2414
B I O C H E M I S T R Y , L’OL.
I 3,
NO.
1
I ?
1974
region. When the modification was performed in the absence of Mg2+ and K- and in the presence of 3 mM EDTA, the binding of the drug increased to 1.6 mol/mol of tRNA. DEAE-cellulose chromatography of a pancreatic ribonuclease hydrolysate revealed, in addition to the previously described modifications, a modified tetranucleotide. The latter was presumably due to modification of a guanosine at position 15, on the 5’ side of the dihydrouridine loop. The amino acid acceptor activity of tyrosine tRNA modified in the presence of Mg*+and K+ was decreased by 18 %; whereas the tyrosine acceptor activity of tRNA modified in the absence of Mgz+and K+ and in the presence of EDTA was decreased to 50 of that of the unmodified tRNA.
useful reagent for chemical modification, since it binds preferentially to the C-8 position of guanosine residues located in single-stranded regions of nucleic acids (Miller et a[., 1966; Kriek et a/., 1967; Fu.1inw-a et a/., 1972; Levine et a/., 1974). In the preceding paper of this series (Fujimura et a/., 1972), we described modification of purified E. coli tRNAflret by reaction with AcN(OAc)Fln, and determined that the primary target in this tRNA was a guanosine residue at position 20 in the dihydrouridine loop. In the present study we describe the effects of the ionic environment on AcN(0Ac)Fln modification of purified yeast tRNATyr. The extent and specific sites of base modification have been determined in a high and in a low ionic strength environment. In addition, the effects of drug modification on aminoacylation and codon recognition of this tRNA are presented. Experimental Section Materials. N-Aceto~y-9-[~~CC]-2-acetylaminofluorene was prepared by the method of Miller et a[. (1966) from N-2hydroxy-9-[ 4C]acetylaminofluorene, which was purchased from the International Chemical and Nuclear Co. The specific activity was 1.264 Cijmol. A 20 mM stock solution of this drug in ethanol was prepared immediately prior to use. Purified yeast tRNATyr was prepared according to Gillam et a / . (1967) from total tRNA purchased from the Plenum Scientific Research Inc. The steps included BD-cellulose chromatography of total tRNA to isolate the tRNATyr rich fraction. This material was then charged with tyrosine and run on a second BD-cellulose column where the charged tRNA now eluted in the ethanol region. The final material accepted approximately 1100 pmol of tyrosine per AL’GI, unit. The triribonucleoside diphosphate U-A-U was prepared by enzymatic (ribonuclease A) synthesis from uridine 2 ’,3 ’-cyclic phosphate and A-U as described by Srinivasan et a/.(1971).
MODIFICATION OF
tRNATyr
BD-cellulose (2@50 mesh) and [ 1 4 C ] t y r ~ ~ (specific ine activity 50 Ci/mol) were purchased from Schwarz/Mann Biochemical. Prior to use for isolation of modified oligonucleotides, the BD-cellulose was washed sequentially with acetone-ethanol (1 :l), 95 ethanol, 30% ethanol in 1.3 M NaCl, and, finally, with 0.02 M NH4HC03.DEAE-cellulose (Cellex-D) (capacity 0.6 mequiv/g) was obtained from Bio-Rad Laboratories. Ribonuclease A and alkaline phosphatase (Escherichia coli) were purchased from Worthington Biochemical Corp., and snake venom phosphodiesterase (Crotalus adamanteus) from Sigma Chemical Co. Aminex-6 was purchased from Bio-Rad Laboratories, Rockville Center, N. Y. Methods. Spectral Methods. Spectrophotometric measurements were made either on a Beckman spectrophotometer, Model DB-G, or a Zeiss spectrophotometer, Model PMQII. Continuous monitoring of eluates from the DEAE-cellulose and Aminex A-6 columns and dihydrouridine determinations were made on a Gilford multiple sample recording spectrophotometer, Model 2000. Reaction of Yeast tRNATvr with AcN(0Ac)Fln. A 6-ml reaction system contained: 140 units of tRNA, 10 mM sodium cacodylate (pH 7.2), 2 mM AcN(0Ac)-9-[ 14C]Fln, and 30% ethanol. As indicated in the individual experiments, the system also contained 3 mM EDTA, 3 mM MgCI2, or 0.1 M KC1. The reaction mixture was incubated for 4 hr at 20” in the dark and then extracted five times with an equal volume of ether. The aqueous phase was adjusted to 0.2 M with potassium acetate (pH 5.0) and precipitated with 2.5 volumes of ethanol and stored at -20”. After centrifugation, the precipitate was dissolved in 2 ml of water. To determine the rate of modification, aliquots were taken after various incubation times, coprecipitated with carrier tRNA, filtered through glass fiber filters, and washed five times with absolute ethanol and five times with ether. The filters were dried, and radioactivity was measured in a NuclearChicago Mark I1 liquid scintillation spectrometer in 5 ml of a toluene scintillation solution. Separation of Modified t R N A from the Unmodified Fraction. Modified tRNA was separated from the unmodified fraction by chromatography on a BD-cellulose column (2 X 5 cm). The column was first equilibrated with 0.01 M sodium acetate (pH 5.0). After the sample (140 units) was applied, the column was washed with 50 ml of the same buffer, followed by 50 ml of 1.3 M NaCl in 0.01 M sodium acetate (pH 5.0). This eluted the unmodified fraction of tRNA, whereas the drug-modified fraction remained bound to the column and was then eluted with 1.3 M NaCl and 30% ethanol in 0.01 M sodium acetate (pH 5.0). Fractions (1.5 ml) were monitored for absorbancy at 260 nm, and the radioactivity of 50-p1 aliquots was assayed in 5 ml of Bray’s solution (Bray, 1960). The unmodified tRNATyr from the “salt region” and the modified tRNAT=’ from the “ethanol region” were pooled separately, dialyzed against water overnight at 4”, and lyophilized. The unmodified material obtained from the “salt region” was used as a control in subsequent assays. RNase A Digestion 0-f Modified tRNA. The mixture (final volume 0.2 ml) contained: 0.02 M Tris-HC1 (pH 7.5), 50 A2e0units of tRNA, and 100 pg of RNase A. Incubation was carried out for 30 min at 37”. Separation of Oligonucleotides by DEAE-Cellulose Chromatography. DEAE-cellulose column chromatography of the RNase A digests of tRNATYr was performed essentially as described by Madison et al. (1967). A column (0.45 x 50 cm) was equilibrated with 0.02 M Tris-HCI (pH 7.0) and 7 M urea. The sample was brought to 7 M with solid urea before applying
it to the column. Oligonucleotides were eluted using a linear 200-ml gradient of 0-0.3 M NaCl in 0.02 M Tris-HCI (pH 7.0) and 7 M urea. Fractions (2 ml) were collected and monitored for uv absorbance at 260 nm. Radioactivity was assayed in 50-p1 aliquots in 5 ml of Bray’s solution. Isolation of Modijied Oligonucleotides on a BD-Cellulose Column. The major radioactive peaks obtained from the above column were pooled, diluted fivefold with water, and applied to a BD-cellulose column (1 X 3 cm) to remove the urea and NaCl and also to separate the modified oligonucleotides from overlapping unmodified ones. The BD-cellulose column was preequilibrated with 0.01 M ammonium bicarbonate and, after the sample was applied, the column was eluted with 50 ml of 0.01 M ammonium bicarbonate, followed by 50 ml of 1.3 M ammonium bicarbonate, and, finally, with 30% ethanol in 1.3 M ammonium bicarbonate. The eluate was monitored with an LKB recording spectrophotometer for uv absorbancy and the fractions were assayed for radioactivity. The first eluates contained some nonradioactive oligonucleotides, whereas the ethanol eluate contained the purified radioactive oligonucleotides. The latter material was pooled, evaporated, suspended in distilled water, and reevaporated to remove all of the ammonium bicarbonate. PuriJcation of Modijied Oligonucleotides by Thin Layer Chromatography ( T k ) . Cellulose plates (Eastman Kodak Co. 6065) were previously washed with water and with isobutyric acid-0.5 M NHIOH (5:3, v/v) and dried, and the samples from the BD-cellulose column were applied. The plates were then developed in the same isobutyric acid-ammonia system. Enzymatic Digestion of Modi3ed Oligonucleotides. The uv absorbing and radioactive spots purified on the tlc plates were removed by scraping, eluted with water, evaporated to dryness on a flash evaporator, and dissolved in 100 p l of 0.2 M ammonium bicarbonate; 10 pg of E . coli alkaline phosphatase and 10 pg of snake venom phosphodiesterase were added to each sample and the mixture was incubated for 16 hr at 37”. Aminex A-6 Column Chromatography. Chromatographic separation of nucleosides was essentially as described by Uziel and coworkers (1968). The Aminex A-6 column (0.45 X 70 cm) was previously washed with 0.4 M ammonium formate (pH 4.7). The above enzymatic digests were applied directly to the column and were eluted with the same buffer. The elution profiles of nucleosides were continuously recorded with a Gilford multiple sample recorder (Model 2000) at 260 nm using a full scale deflection of 0.15 Anso.The identification of nucleosides was provided by their elution position and by their uv spectra. The amount of a nucleoside was determined by measuring its A260 absorbancy area with a planimeter and then comparing this to the area obtained with known amounts of authentic samples. Dihydrouridine Assay. Dihydrouridine was determined both qualitatively and quantitatively on an aliquot of the sample by observing the loss of absorbancy at 235 nm in 0.02 M NaOH according to Batt and coworkers (1954). The method was applied directly to samples obtained from the Aminex A-6 column. Assay of Tyrosine Acceptor Actiaity. Rat liver aminoacyltRNA synthetase was prepared from the 105,OOOg supernatant fraction by protamine sulfate precipitation, ammonium sulphate precipitation (70% of saturation at 20”), and dialysis against 50% glycerol, 0.01 M Tris-HCI (pH 7.5), 0.06 M KCI, 0.01 M Mgz+, and 0.05 M mercaptoethanol, as previously described by Nishimura and Weinstein (1969). The assay system contained in a total volume of 100 pl: 0.01 M potassium cacodylate (pH 7.5), 0.01 M KCI, 0.016 M MgC12, 2 mM ATP, 0.2 pCi of BIOCHEMISTRY VO , L. 13,
NO.
11, 1 9 7 4
2415
PULKRABEK,
GRUNBERGER,
A N D WEINSTEIN
Effects of Ionic Conditions on Extent of AcNH-Fln Modifications and Tyrosine Acceptor Activity of Yeast tRNATyr.
TABLE I:
2 -
r.3
'0 X
z a o
Moles of AcNH-Fln Tyrosine Acceptor Bound/Mol Activity Expressed of tRNA as of Control'
I -
Modified in 3 mM MgClz-0.1 3 mM MgC12 3 mM EDTA
TIME IHRI FIGURE
1: Kinetics of the reaction of yeast tRNATsr with AcNH-
(Ac0H)Fln under different ionic conditions. Incubation mixture (0.3 ml) contained 5 units of yeast tRNATrTin 10 mM sodium cacodylate (pH 7.2), 2 mM [14C]AcN(OAc)Flnin 33% ethanol, and indicated amounts of MgCI,, KCI, or EDTA. Incubation was carried out at 37", and 50-pl aliquots were precipitated with 10% trichloroacetic acid at indicated time intervals and processed as described in Methods: (0)3 mM MgCl,-O.l M KCI; ( 0 )3 mM MgCL ( 0 )3 m M EDTA. [14C]tyrosine, 0.05 '4260 of tRNA, and about 0.05 mg of crude aminoacyl-tRNA synthetase. The reaction mixture was incubated at 37" for 15 min, precipitated with 3 ml of 5 % trichloroacetic acid, filtered through Millipore membrane filters (type HA), and washed three times with 3 ml of 5 trichloroacetic acid. Radioactivity was measured in 5 ml of toluene scintillation solution. Ribosomal Binding Assay. The ribosomal binding of [ tyrosyl-tRNA stimulated by the triplet U-A-U was carried out according to Nirenberg and Leder (1964). The incubation mixture (final vol 50 pl) contained: 0.01 M Tris-HC1 (pH 7.2), 0.01 M ammonium acetate, 0.02 M MgCI?, 1.7 A260units of E . coli ribosomes, approximately 125 pmol of [14C]tyrosyltRNATYr, and 0.15 A260 unit of U-A-U. Incubation was carried out at 24" for 20 min. The reaction mixture was diluted with 3 ml of cold 0.1 M Tris-HC1 (pH 7.2) containing 0.01 M Mg*+ and 0.1 M ammonium acetate, filtered on a Millipore membrane (type HA), washed three times with 5 ml of cold buffer, and assayed for radioactivity.
E
2.0j
0
5
10 15 20 2 5 F R A C T I O N NUMBER
30
2: Separation of yeast tRNAT?' modified with [I4C]AcN(Ac0H)Fln from unmodified tRNATrf'on a BD-cellulose column. The reaction mixture contained in a total volume of 6 ml: 140 A260 units of tRNATsr, 2.0 mM [14C]AcN(OAc)Fln, I O mM sodium cacodylate (pH 7.2), and 33 ethanol. Incubation was carried out for 4 hr at 37" in the dark; samples were extracted five times with ether, and the tRNA in the aqueous phase was precipitated with 2.5 vol of ethanol at -20" and finally dissolved in 2 ml of water. The sample was applied to a BD-cellulose column (2 X 5 em) and eluted as described in Methods. FIGURE
2416
B I O C H E M I S T R Y , VOL.
13,
NO.
11, 1 9 7 4
M
KCI
1.08 1.20 1.63
81 Not done 50
a The data were obtained on AcNH-Fln-modified tRNA which was previously separated from the unmodified fraction on a BD-cellulose column. Reaction conditions and processing of the samples are described in Methods. A per cent inhibition is related to the acceptor activity of an equivalent amount of unmodified tRNATyr taken from the 1.3 M region of a BDcellulose column.
Results Efects of Salts on Extent of Modification of Yeast tRNATu' with N-Acetoxy-2-acetylaminof7uorene.The time course of tRNATYrmodification in the presence of either 3 mM EDTA, 3 mM MgCl?, or 3 mM MgCl? and 0.1 M KC1 is shown in Figure 1. In all cases maximum binding was reached after about 4 hr of incubation. To calculate the exact number of radioactive acetylaminofluorene moieties bound per molecule of tRNA, it was necessary to separate the modified tRNA molecules from those that might have escaped modification. This was achieved by fractionating the tRNA obtained from the reaction mixture on a BD-cellulose column (Figure 2). As demonstrated previously (Fink et al., 1970; Fujimura et al., 1972) with other tRNAs, the unmodified tRNA molecules were eluted with 1.3 M NaCl whereas the modified tRNA molecules, due to the hydrophobic character of the carcinogen, were eluted only with a mixture of 30% ethanol and 1.3 M NaC1. In all three reaction conditions about 50% of the tRNATYr molecules were modified. The different ionic conditions did, however, influence the specific activity of the drugmodified fraction. Table I indicates that in the presence of 3 mM MgC12 and 0.1 M KCl only 1.08 AcNH-Fln residues were bound per molecule of tRNA. When 0.1 M KC1 was omitted from the reaction mixture the ratio of drug to tRNA increased to 1.20. When both 3 mM MgClz and 0.1 M KCl were omitted and 3 mM EDTA was added to the reaction mixture the modification ratio increased to 1.63. Under these experimental conditions not necessarily all of the stabilizing cations present in the "interior" of the tRNA molecule were removed (Goldstein et al., 1972). RNase A Hydrolysis and Location of Modified Guanosine Residues in tRNATy'. Preparations of yeast tRNATyr reacted with AcN(0Ac)Fln in the presence of MgCh and KC1 or in the absence of MgCh and KC1 and, in the presence of EDTA, were digested with pancreatic ribonuclease, and the oligonucleotides were separated on a DEAE-cellulose column with 7 M urea and a NaCl gradient (Figures 3 and 4). Eight uv absorbing peaks were reproducibly obtained, and the profile was similar to that previously described by Madison et al. (1967). The fact that only two or three radioactive peaks were obtained (see below) indicates that the drug specifically modifies only a few nucleoside residues in yeast tRNATyr and does not attack randomly.
MODIFICATION OF
tRNATYr
i
z
0 UY
A 4
2-
% W
u z 4
m
IT
8m 1 4
m
E
0
m l
9
0
20
IO
30
40 50 60 70 FRACTION N U M B E R
80
90
FIGURE
3 : DEAE-cellulose column chromatography of a RNase A hydrolysate of modified yeast tRNATyr. Fifty units of tRNATrr modified with [14C]AcNH-Flnin 3 mM MgCI, and 0.1 M KCl were hydrolyzed with RNase A and chromatographed in a DEAE-cellulose column as described in Methods.
DEAE-cellulose column chromatography of a RNase A hydrolysate of modified yeast tRNATyr. Fifty Azsounits of tRNATyr modified with [14C]AcNH-Flnin 3 mM EDTA were hydrolyzed with RNase A and chromatographed on a DEAE-cellulose column as described in Methods.
In the hydrolysate of tRNA modified in the presence of 3 mM MgCln and 0.1 M KC1, only two significant radioactive peaks were observed and these occurred in a ratio to each other of approximately 1 : 4 (Figure 3). The smaller peak was in the region of dinucleotides (fractions 42-48, Figure 3), and the major one in the region of trinucleotides (fractions 52-58, Figure 3). The oligonucleotides containing bound drug actually elute somewhat later from the DEAE-cellulose column than the corresponding unmodified materials. This is consistent with previous evidence that oligonucleotide containing AcNH-Fln have a higher affinity for DEAE-Sephadex (Fujimura et a/., 1972). In the hydrolysate of tRNA modified in the presence of EDTA, three major radioactive peaks were obtained ; one in the region of dinucleotides (fractions 44-52, Figure 4); one in the region of trinucleotides (fractions 56-62, Figure 4), and the third in the region of tetranucleotides (fractions 67-76, Figure 4) were pooled and separated from unmodified oligonucleotides on BD-cellulose columns as described in Methods. The major radioactive peak obtained from tRNA modified in the presence of MgClz and KC1 (Figure 3) was collected and further purified on a BD-cellulose column and by thinlayer chromatography (see Methods). Three uv spots were detected on tlc but only one of these (RF0.38) was both fluorescent and radioactive. The two other spots were nonradioactive. The base composition of the radioactive spot was obtained by complete digestion with snake venom phosphodiesterase and E. coli alkaline phosphatase and nucleoside analysis on the Aminex A-6 column. The results of the chromatographic separation are shown in Figure 5. Guanosine and 2'-O-methylguanosine (2'-MeGuo) in a ratio of approximately 2 : l were detected. Evidence for the presence. of dihydrouridine in the same oligonucleotide was provided by a sharp loss of absorption at 235 nm after alkalinization of either an aliquot of the digested oligonucleotide or of the fractions which were eluted in the uridine region of the Aminex A-6 column (Figure 5). None of the radioactivity applied to the column appeared in the eluate. This is consistent with separate studies indicating that AcNH-Fln modified guanosine residues are bound extremely tightly to Aminex A-6.
The primary structure of yeast tRNATyr, established by Madison and Kung (1967), is shown in Figure 6. Since the base composition of the major AcNH-Fln modified oligonucleotide indicated the presence of Guo, 2'-MeGuo, and H2Urd, we concluded that this modification occurred in the dihydrouridine loop in the region 18-20 (Figure 6). Since both free Guo and 2'-MeGuo were obtained in the hydrolysate of the purified AcNH-Fln modified triplet, it appears that the drug had modified in certain molecules of tRNA Guo at position 18 whereas in other molecules 2'-MeGuo modification occurred at position 19. In separate studies we were able to demonstrate that AcN(0Ac)-Fln reacts with 2 '-MeGuo nucleoside as well as with Guo. The amount of material present in the minor peak located in the dinucleotide region (fractions 42-47, Figure 3) and in the tetranucleotide (fractions 67-76, Figure 4) was in-
FIGURE 4:
.
IO
0 (D
2-05
STANDARDS
2'MeGuo
A
STANDARD
.I 0
i
SAMPLE
0 F: .O
2Urd SSAY
2 .o FRACTION NUMBER
5: Aminex A-6 column chromatography of nucleosides. Purified radioactive oligonucleotide was digested with snake venom phosphodiesterase and E . coli alkaline phosphatase, and applied on an Aminex A-6 column (0.45 X 70 cm). The elution profile was compared with that obtained with authentic nucleosides. Dihydrouridine was assayed in fractions obtained from the column as described in Methods.
FIGURE
B I O C H E M I S T R Y , VOL.
13,
NO.
11, 1 9 7 4
2417
P U L K R A B E K,
G R U N B E R G E R,
A N D
W E 1N S T EI N
UAU Stimulated Binding of Tyrosyl-tRNA to Ribosomes."
TABLE 11:
A 1-pC*G
U4A
[ 14C]Tyrosyl-
C*G
tRNATyr Bound
U*G
C * G-70 G*C CG * a GC
G* G* AA YDD
30-A* U A * U Gm
C
A*W
Control AcNH-Fln-modified
5.22 3.05
Apmol 100 58
19.43 15.99
100 82
A-40 IA G*,, A
u
FIGURE 6 : Primary structure of yeast tRNATrr with probable sites of AcNH-Fln modification. The primary structure of tRNATsr is taken from Madison et at. (1967): (*) apparent sites of AcNHFln modification in the native form: C*)additional site of modification in the denatured form.
sufficient to determine their base composition. It is likely, however, that the dinucleotide was derived from the G-$ sequence (positions 36 and 37; Figure 6), and the tetranucleotide from the A-A-G-D (positions 13-16, Figure 6); see Discussion. Tjmsine Acceptor Acticity o f Modijied Yeast t R N A T y r . The tyrosine acceptor activity of yeast tRNATYr which had been reacted with AcNH(0Ac)Fln in a medium of either high or low ionic strength is given in Table I. In these studies the modified molecules were first separated from the unmodified fraction by the BD-cellulose procedure and their acceptor activities were then compared. It can be seen that the tRNA modified with drug in the presence of MgCl? and KCl retained S1 % of the acceptor activity of an equivalent amount of unmodified tRNA. The tRNA modified in the absence of MgCI? and KC1 and in the presence of EDTA retained only 5 0 z of the acceptor activity of the control material, presumably reflecting the greater extent of modification (Table I). It is possible that the 19% decrease in the case of tRNA modified in the presence of MgCI? and KCI relates to that fraction of molecules which were modified in the anticodon. This explanation is consistent with the quantitative relations of the two peaks seen in Figure 3, although other explanations cannot be excluded. Codon Dependent Binding of Modified t R N A to Ribosomes. In Table I1 is shown U-A-U-stimulated binding of [l4Cc]tyrosyl-tRNA t o ribosomes. Results obtained with control tRNA are compared to those obtained with tRNA previously modified with the drug in the presence of MgCh and KCI. The binding to ribosomes of the [14C]tyrosyl-tRNAmodified with AcNH-Fln was only 58% of that obtained with the unmodified tRNA. In part, this could reflect the fact that the modified sample was less fully charged with [14C]tyrosinethan the control sample, since there is evidence that interaction with codon and ribosomes takes place with nonacylated tRNA as well as aminoacylated tRNA (Levin and Nirenberg, 1968; Levin, 1970). A correction for the difference in specific activities of the two tRNAs was therefore made to calculate BIOCHEMISTRY,
VOL.
a Reaction mixtures contained 125 pmol (as bound tyrosine) of either control or AcNH-Fln-modified [ 14C]tyrosyl-tRNA. Their specific activities were 430 and 350 pmol of [ 14C]tyrosine/ A260of tRNA, respectively. The AcNH-Fln-modified tRNA had been reacted with the drug in the presence of MgCI2 and KCI and is the same sample as that in line 1, Table I. Assays and the processing of the samples were as described in Methods. [14C]tyrosyl-tRNA bound is the amount of tyrosyl-tRNA bound in the presence of UAU minus the amount bound in the absence of UAU. 'Total tRNATyr bound is a derived value which corrects for the differences in specific activities of modified and unmodified tRNAsTyr, assuming that both the aminoacylated as well as the nonacylated tRNAs bind to ribosomes.
'
C
2418
Apmol
50
m2'
20
TypeoftRNAAdded
Total tRNATyr Bound'
13,
NO.
11, 1 9 7 4
the total amount (both acylated and nonacylated) of tRNA'rYr bound to ribosomes. Based on this assumption, the binding of AcNH-Fln modified tRNA was only 18% less than that obtained with the control tRNA (Table 11). This decreasc corresponds approximately to our estimate of the fraction of tRNA molecules which were modified in the dinucleotide present in the anticodon of this tRNA (see above and Discussion). Discussion Based on data obtained from chemical modification studies and physical measurements, several models have been proposed for the secondary and tertiary structures of tRNA (for a review, see Cramer, 1971). Recently the tertiary structure of yeast phenylalanine tRNA was determined by X-ray crystallography studies (Kim et al., 1973). It is of interest, therefore, to analyze our results in terms of previous proposals for the structure of tRNA. N-2-Acetylaminofluorene is a particularly interesting probe for analyzing the secondary and tertiary structures of tRNA in solution because of its preferential interaction with G residues in single-stranded regions of nucleic acids (Fujimura et al., 1972; Levine et al., 1974). According to the cloverleaf model of yeast tRNATYr (Figure 6), 16 out of 23 guanosine residues are in doublestranded regions. Several of the proposed tRNA models suggest an additional base pair between G-15 and the C residue (C-58) in the sequence T-Q-C (Cramer et al., 1968, 1969). This would diminish the probability of interaction of AcN(OH)-Fln with either G-15 or G-59. Previous studies indicated that residues in the dihydrouridine loop are readily attacked by a variety of chemical agents (Fujimura et al., 1972; Cramer et al., 1968; Igo-Kemenes and Zachau, 1969; Litt, 1969; Chang et al., 1972). The data obtained in the present study indicate that residues 18 and 19 in the dihydrouridine loop of yeast tRNATyr are very susceptible to AcNH-Fln modification, suggesting that in this respect the tertiary structure of tRNATYris similar to that of previously studied tRNAs.
MODIFICATION OF
tRNATyr
The anticodon regions of tRNAs should also be relatively exposed since they must interact with codons in mRNA. Kethoxal modification of a guanosine residue in the anticodon loop of yeast tRNAPhehas been described (Litt, 1969; Litt and Hancock, 1967). Other residues in the anticodon region of tRNAs are susceptible to chemical modification by borohydride (Igo-Kemenes and Zachau, 1969) and monoperphthalic acid (Cramer et al., 1968). In the RNase A digest of AcNH-Fln modified yeast tRNATyr we observed, in addition to the major modification described above, lesser amounts of an AcNH-Fln containing dinucleotide. We have been unable to obtain sufficient amounts of this material for direct analysis of its base composition. There are, however, only three dinucleotides which have the general structure G-Y in an RNase A digest of yeast tRNATyr: (1) mz2G-C (positions 28 and 29); (2) G-T (positions 55 and 56), and (3) G-P (positions 36 and 37). Modification of Mz2G-C is unlikely since it is sterically buried and thus far no chemical modifications of this region have been reported. G-55 is probably base paired with C-63 (see above) and, therefore, would not be readily susceptible to modification. We conclude, therefore, that the AcNH-Fln containing dinucleotide probably represents modification of G-36 which is present in the anticodon of tRNAT”’. The decreased activity of AcNH-Fln modified [ * 4C]tyrosyl-tRNA in a codon stimulated ribosomal binding assay is consistent with this interpretation, and it is known that AcNH-Fln modification of G residues in oligo- or polynucleotides impairs their basepairing capacity (Grunberger et al., 1970; Grunberger and Weinstein, 1971; Levine et al., 1974). On the other hand, we have not excluded the possibility that the decrease of the acceptor activity as well as that of the ribosomal binding could be due to an overall conformational change in the modified tRNA. Exposure of tRNATyr t o AcN(0Ac)Fln in a low rather than a high ionic strength environment resulted in at least one additional site of drug modification. After RNase A digestion this modification was recovered in a tetranucleotide. Only two tetranucleotides containing G are present in a RNase A digest of yeast tRNATyr (Madison et al., 1967, and Figure 6), A-A-G-D (positions 13-16) and G-G-G-C (positions 51-55). AcNH-Fln modification of the former is most likely to occur because: (1) the latter sequence is in a double-stranded region, and (2) in a low ionic environment, partial unfolding of the tRNA molecule would be associated with a loss of the G-15 to C-58 base pair, thus rendering G-15 susceptible to chemical modification. Streeck and Zachau (1972) found that with denatured yeast tRNAPheTz RNase produced a split in the dihydrouridine loop at G-15 which did not occur with native tRNAPhe,suggesting that denaturation does selectively unfold this region of the tRNA molecule. Although additional studies are required to more firmly establish each of the sites of AcNH-Fln modification in tRNATyr, it is apparent that the modification is quite specific and sensitive to the conformation of the tRNA molecule. This drug should prove to be a valuable tool for further exploring the various conformations of a single species of tRNA as a function of the solvent and ionic environment. From the biological point of view, it may also be of interest that the present results suggest that the extent and specific sites of nucleic acid modification by this carcinogen are a function of the ionic environment.
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NO.
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